Gli autori vorrebbero riconoscere il finanziamento del Ministero della Pubblica Istruzione ACRF Tier 1 (RG41) e l&#39;avvio di sovvenzione da Nanyang Technological University. Basso e Tjin sono finanziati dalla borsa di ricerca per studenti (RSS) da Nanyang Technological University, Singapore.

1. Clonazione di ricombinante plasmidi codifica per proteine ​​AECM <ol><li> Progettare la sequenza aminoacidica del dominio funzionale (ad esempio, cellule dominio vincolanti e ripete elastina-like). Siti di restrizione design che fiancheggiano le estremità dei domini funzionali per facilitare sub-clonazione usando il software libero secondo le istruzioni del software (ad esempio, http://biologylabs.utah.edu/jorgensen/wayned/ape/). Qui, selezionare i siti unici di restrizione che non sono presenti nel dominio funzionale di confinare la digestione ai siti previsti. Scegliere siti di restrizione che appaiono nel sito di clonaggio multiplo (MCS) del vettore host (ad esempio, pET22b (+)) per sub-clonazione. </li><li> Reverse sequenza di amminoacidi nella sequenza nucleotidica utilizzando siti gratuiti secondo le istruzioni del software. (Ad esempio, http://www.bioinformatics.org/sms2/rev_trans.html). Assicurarsi codoni sono ottimizzati per E. host coli. </li><li> Gene di interesse può essere purchasoligonucleotidi incagliati ed in commercio come singoli (cioè, se gli oligonucleotidi &lt;100 coppie di basi (bp)) e permette di effettuare la ricottura del DNA (vedi punto 1.4) per ridurre i costi. In caso contrario, per i geni più grandi di 100 bp, potrebbero essere acquistati attraverso società commerciali. </li><li> Ricottura DNA degli oligonucleotidi <ol><li> Ricuocere gli oligomeri di DNA per ottenere la sequenza del gene desiderato. Sciogliere oligonucleotidi di DNA in un buffer oligomero DNA (Tris 10 mM: 2-ammino-2- (idrossimetil) propan-1,3-diolo, pH 8,0, filtrato) ad una concentrazione finale di 1 mg / mL. </li><li> Aggiungere 4 ml di ogni oligomero di 32 microlitri di tampone di ricottura DNA (Tris 10 mM, NaCl 100 mM e 100 nM MgCl 2) per ottenere una miscela totale 40 microlitri. </li><li> Far bollire un bicchiere di acqua con una piastra e immergere la miscela per 5 minuti, 95 ° C. Togliere il bicchiere e gradualmente raffreddare l&#39;intero set in una scatola di polistirolo O / N. Gli oligomeri sono stati ricotti e sono pronti per la digestione. </li></ol></li&gt; <li> Aggiungere i corrispondenti enzimi di restrizione per digerire gli oligomeri ricotto (cioè, denominato insert) e vettore ospitante (cioè, pET22b (+)) separatamente. Utilizzare la seguente ricetta (1-2 mg di DNA, 2 microlitri di ciascun enzima di restrizione, 5 ml di 10x tampone di restrizione enzimatica, e acqua fino a una miscela totale 50 microlitri) per 3-4 ore a 37 ° C. NOTA: Il pET22b (+) vettore plasmidico è ampicillina resistenti agli antibiotici e contiene un 6x-His tag al C-terminale. Il 6x-His tag presenti in pET22b (+) ci permette di usare il suo-tagged anticorpi per identificare la proteina bersaglio mediante western blotting al punto 7. </li><li> Aggiungere caricamento 6x colorante per ogni miscela di digestione. Eseguire le miscele di digestione compreso separatamente una scaletta DNA su 1,2% gel di agarosio DNA contenenti una macchia DNA fluorescente agli UV per 1 ora a 100 V. I dettagli del 1,2% DNA gel di agarosio con un illuminatore luce UV. </li><li> Tagliate il gel per estrarre prodotti di DNA digeriti utilizzando kit di purificazione gel commerciale. Elute utilizzando il volume minimo della colonna per ottenere una concentrazione minima di DNA di 50-100 ng / ml. </li><li> Combinare il gene di interesse sequenzialmente legando l&#39;inserto di DNA digerito (cioè, ripetizioni elastina o domini di legame cellulari ottenute mediante ricottura DNA) nel vettore plasmidico utilizzando T4 ligasi utilizzando la seguente ricetta: (2 ml di vettore, 1 ml di T4 ligasi, 1,5 ml di T4 tampone legatura, x ml di inserto, da 10,5 x ml di acqua per una miscela totale 15 ml). Incubare la miscela legatura a temperatura ambiente per 2 ore. NOTA: concentrazione molare di vettore inserire deve essere variato per ottimizzare l&#39;efficienza legatura. Volume di inserto descritto come x nella ricetta dipende dalla concentrazione di DNA eluito dal punto 1.7. </li><li> Thaw E. cellule chimicamente competenti coli DH5α (o qualsiasi sforzo di clonazione) su ghiaccio. Piastre 2xYT Warm agar (Tabella 1) contenente ampicillina (25 mg / ml) a 37 ° C. </li><li> Trasformare le cellule utilizzandoshock termico: <ol><li> Aliquota 50 ml di cellule competenti in provette micro-centrifuga pulite, pre-refrigerati. Pipettare 5 microlitri (tra 100 pg a 100 ng) della miscela di ligasi nelle cellule pipettando delicatamente su e giù per miscelare. Lasciare la miscela in ghiaccio per 20 min. </li><li> Immergere il tubo micro-centrifuga contenente la miscela di cellule in un bagno d&#39;acqua 42 ° per 2 minuti e tornato in ghiaccio per 2 min. Tempo la durata di immersione per minimizzare i danni del calore alle cellule. </li><li> Aggiungere 500 pl di supporti SOC (Tabella 1) nel tubo micro-centrifuga e incubare a 37 ° C con agitazione per 1 ora. </li><li> Distribuire 50 500 microlitri della miscela di cellule / legatura su una piastra di agar 2xYT che è stato pre-riscaldato a RT, contenente ampicillina (25 mcg / ml) e incubare le piastre capovolta a 37 ° CO / N (12-16 hr) . </li></ol></li><li> Il giorno dopo, raccogliere le colonie di DNA dalla piastra di agar con puntali puliti. Far crescere le colonie in 5 ml di 2YT supporti (Tabella1) contenente ampicillina (25 mg / ml) O / N a 37 ° CO / N (12-16 ore) con agitazione (225 rpm). </li><li> Il giorno seguente, utilizzare un kit di isolamento plasmide per estrarre i plasmidi di DNA per ogni colonia raccolti secondo il protocollo del produttore. Eluire il DNA con 50 ml di acqua. </li><li> Eseguire un test digest con enzimi di restrizione utilizzando la seguente ricetta: (5 microlitri DNA, 0,2 ml di ogni enzima di restrizione, 1 ml 10x tampone di restrizione enzimatica e acqua rabboccare per una miscela totale 10 microlitri), incubare per 2 ore a 37 ° C a schermo per le colonie che probabilmente contengono l&#39;inserto e corsa su 1,2% gel di agarosio DNA come al punto 1.6. NOTA: Dopo la digestione, una legatura di successo dovrebbe risultino due bande, una per ogni vettore e l&#39;inserto che corrispondono ai rispettivi peso molecolare (Figura 1). </li><li> Invia le colonie probabili per il sequenziamento del DNA usando promotore T7 avanti e indietro primer per sequencer commerciali. </li></ol><ol><li> Dal risultato sequenziamento, selezionare una colonia che è stato ligato con successo e utilizzare il DNA plasmidico per trasformazione in E. ospite espressione coli. </li><li> Thaw E. ceppo espressione coli (BL21 (DE3) pLysS) su ghiaccio. Nel frattempo, piastre di agar caldi contenenti ampicillina (25 mcg / ml) e cloramfenicolo (34 mg / ml) per RT. NOTA: Il plasmide pLysS presente nei batteri ceppo BL21 (DE3) pLysS contiene un gene di resistenza cloramfenicolo. Il plasmide pLysS contiene un repressore gene T7, che è costitutivamente espresso per limitare l&#39;espressione della proteina che perde AECM. Cloramfenicolo è necessario selezionare per cellule batteriche contenenti pLysS durante la coltura. </li><li> Ripetere il punto 1.10 di ottenere trasformato E. coli che sono pronti a esprimere le proteine ​​artificiali. Avvolgere piastre di agar in Parafilm e conservare a testa in giù a 4 6; C per un massimo di un mese. </li></ol> 3. L&#39;espressione batterica di AECM Proteine <ol><li> Vedere la Figura 2, scegliere una colonia dalla piastra di agar trasformata con un puntale e inoculare in 10 ml di sterili Terrific Broth (TB) i media (Tabella 1) che contiene sia ampicillina e antibiotici cloramfenicolo in una provetta. Incubare questa coltura starter a 37 ° CO / N (12-16 ore) con agitazione a 225 rpm. </li><li> Trasferimento 10 ml di coltura starter in 1 L mezzi TB freschi e sterili integrate con gli stessi antibiotici in un pallone 3 L Erlenmeyer. Incubare la coltura a 37 ° C con agitazione a 225 rpm per 2-3 ore ed osservare la densità ottica della coltura (OD 600) raggiunto 0,6-0,8 pipettando 1 ml di coltura in una cuvetta vuota per la lettura. Salva 1 ml di coltura prima dell&#39;induzione per SDS-PAGE caratterizzazione. <ol><li> Per misurare OD 600 della coltura cellulare, preparare 1 fiala di mezzi TB in una provettaper una misurazione vuoto. Separatamente, trasferire 1 ml di coltura dal pallone di coltura in una nuova cuvetta vuota. Misurare la densità ottica della coltura utilizzando uno spettrofotometro contro il controllo in bianco in assorbanza a 600 nm. </li><li> Salvare il campione (per la successiva analisi SDS-PAGE) trasferendo i 1 ml di campione di coltura in una provetta da 1,5 ml microcentrifuga, centrifugare a 12.000 xg per 2 minuti e decantare il surnatante. </li></ol></li><li> Indurre la coltura con isopropil β-D-1-thiogalactopyranoside (IPTG) ad una concentrazione finale di 1 mM e incubare a 37 ° C con agitazione a 225 rpm per altri 4 ore. Salva 1 ml di coltura alla fine dell&#39;induzione a 4 ore per SDS-PAGE caratterizzazione. </li><li> Raccogliere le cellule trasferendo cultura per bottiglie da centrifuga 1 L e centrifugare a 12.000 xg per 30 min a 4 ° C. Eliminare il surnatante, pesare il pellet di cellule e risospendere in TEN tampone (1 M Tris, 0,01 M EDTA, 0,1 M di NaCl, pH = 8.0) a 0,5 g / ml. </li></ol> 4. Lisi di colture batteriche <ol><li> Congelare la coltura cellulare risospeso a -80 ° CO / N. Scongelare la coltura cellulare congelato in un bagno d&#39;acqua a temperatura ambiente o su ghiaccio per lisare le cellule. Aggiungere 10 ug / ml Deoxyribonuclease I (DNasi I), 10 mg / ml ribonucleasi A (RNasi I), e 50 ug / ml phenylmethylsulfonyl fluoruro (PMSF) mentre scongelamento e omogeneizzare la soluzione con lenta agitazione. </li><li> Dopo che tutte le cellule risospese vengono scongelate, regolare la soluzione a pH 9,0 per aumentare la solubilità della proteina in acqua 12. Aggiungere 6 N NaOH goccia a goccia sotto agitazione su ghiaccio per ottenere una consistenza omogenea. Lyse dalla rottura ultrasuoni per 20 minuti sul ghiaccio con una punta piatta 2 mm di diametro, 5 sec impulso. </li><li> Centrifugare la soluzione di cellule a 12,000 xg per 30 min a 4 ° C. Trasferire il surnatante di un ambiente pulito, una bottiglia vuota e conservare a 4 ° C per la purificazione dopo. </li><li> Nel frattempo, risospendere il pellet cellulare nuovo con TEN tampone e ri-congelamento a -80 ° C. Alisi cellulare completo, ripetizione gelo / disgelo e sonicazione processo fino a tre volte. Risparmia il 20 lisato cellulare microlitri per SDS-PAGE caratterizzazione. </li></ol> 5. Purificazione di all&#39;AECM proteine ​​Utilizzando Inverse transizione in bicicletta <ol><li> Fascicola lisato cellulare dal punto 4.3 e procedere per purificare le proteine ​​all&#39;AECM utilizzando ITC, simile a quello utilizzato per la purificazione di proteine ​​di elastina-21. Cicli saranno fatte con diverse temperature che sono di 4 ° C (indicati come &quot;freddo&quot;) e 37 ° C (denominati &quot;caldi&quot;), cicli. </li><li> Dividere il lisato cellulare in 50 ml bottiglie centrifuga e centrifugare a 40.000 xg per 2 ore a 4 ° C. L&#39;aspetto del pellet deve essere di colore marrone scuro e guardare che cola. </li><li> Rimuovere il surnatante pipettando per ottenere una netta separazione dal pellet. Raccogliere il supernatante in bottiglie da centrifuga puliti e aggiungere NaCl ad una concentrazione finale di 1 M (per un massimo di 3 M). Caldola soluzione a 37 ° C per 2 ore con agitazione a 225 rpm. NOTA: L&#39;aggiunta di NaCl attiverà il passaggio della componente elastina dell&#39;aggregazione causando AECM. Il surnatante diventerà torbido. Se la concentrazione proteica è sufficientemente elevata, proteine ​​schiumoso bianco può essere visto ai lati della bottiglia centrifuga. </li><li> Centrifugare il surnatante dal punto 5.3 a 40.000 xg per 2 ore a 37 ° C. Decantare il surnatante. Schiacciare il pellet con una spatola metallica e risospendere le bit pellet in acqua distillata autoclavata ghiacciata (50 mg / ml) con agitazione vigorosa O / N a 4 ° C utilizzando una ancoretta magnetica e la piastra. Il pellet dovrebbe essere completamente sciolto. </li><li> Ripetere i passaggi 5,2-5,4 per altri tre a cinque volte per ottenere una maggiore purezza della proteina. </li><li> Dopo il ciclo finale di purificazione, desalificare la soluzione proteica da dialisi contro acqua distillata a 4 ° C. Dializzare soluzione proteica contro l&#39;acqua per 2-3 giorni con i cambiamenti in acqua ogni 4hr o 8 ore per O / N. Invia 20 microlitri della proteina purificata per SDS-PAGE e liofilizzare il resto della proteina purificata e conservare a -80 ° C fino all&#39;utilizzo. </li></ol> 6. Caratterizzazione di all&#39;AECM proteine ​​Utilizzando SDS-PAGE elettroforesi <ol><li> Preparare un SDS-PAGE gel al 12% (se il peso molecolare è di grandi dimensioni, utilizzare 8% gel SDS-PAGE per una migliore separazione). Aggiungere 2x tampone di caricamento SDS a ciascun campione, riscaldare i campioni per 10 min a 100 ° C ed eseguire campioni sul gel per 1 ora a 100 V o finché la scala della proteina corrispondente al peso molecolare della proteina all&#39;AECM raggiunge la metà del gel. </li><li> Recuperare il gel dalla SDS-PAGE configurare e aggiungere Coomassie Brilliant macchia blu (Tabella 2) con un volume di immergere il gel per 1 ora in una capsula di Petri. Gel risciacquo in una soluzione decolorante (Tabella 2) per 5 minuti su un agitatore meccanico. Cambiare la soluzione decolorante, e continuano a Decolorare il gel con dondolo fino al fondo della tegli gel diventa chiaro. Bande di proteine ​​devono essere chiaramente visibili. </li><li> Confrontare la posizione della proteina bersaglio contro la scala di proteine ​​per determinare il peso molecolare della proteina bersaglio. </li><li> Ad ulteriore conferma della presenza della proteina bersaglio, effettuare western blotting come al punto 7. </li></ol> 7. Caratterizzazione di all&#39;AECM proteine ​​Utilizzando Western Blotting <ol><li> Eseguire i campioni su un SDS-PAGE gel al 12%, come nel capitolo 6, senza colorazione. </li><li> Tagliare membrana di nitrocellulosa a una dimensione leggermente più grande del gel SDS-PAGE. Tagliare 4 pezzi di carta da filtro per le stesse dimensioni. </li><li> Wet un pezzo di carta da filtro con Transfer occidentale Buffer (20% v / v di metanolo, 25 mM Tris, 190 mM glicina, pH 8,3), collocare l&#39;SDS-PAGE gel sulla parte superiore della carta da filtro, seguito da un altro foglio di carta da filtro , quindi la membrana di nitrocellulosa e una finale altre due pezzi di carta da filtro. Garantire l&#39;intero set-up è sommersa con sufficiente buffer di trasferimento occidentale. NOTA: Il gel e membrane non devono essere in contatto, a questo punto di tempo come proteine ​​potrebbe legarsi alla membrana a contatto. </li><li> Trasferire le proteine ​​sulla membrana di nitrocellulosa posizionando due filtri di carta in una unità occidentale trasferimento semi-dry, seguita dalla membrana di nitrocellulosa, e posizionare accuratamente il gel SDS-PAGE all&#39;interno e sulla membrana infine posizionare gli ultimi due carta da filtro umida sulla il gel SDS-PAGE. Eseguire il trasferimento occidentale a 45 mA, 30 min. Aggiungere tampone se la tensione è superiore a 30 V. NOTA: Preferibilmente posizionare la SDS-PAGE gel sulla membrana con un tentativo e evitare di spostare il gel inutilmente sulla membrana come proteine ​​potrebbe legarsi alla membrana a contatto. </li><li> Recuperare e bloccare la membrana di nitrocellulosa con una soluzione bloccante (5% latte scremato in PBS pH 7.4, filtrata con carta da filtro) per 2 ore a temperatura ambiente. Smaltire tampone di bloccaggio e aggiungere PBS per sciacquare. NOTA: Passare nuovi guanti per evitare di contaminare la membrana con prote indesideratiins. </li><li> Incubare primario anti-anticorpo con la sua diluizione in PBS a 1: 1.000 a temperatura ambiente per 1 ora con dondolo. Preparare una miscela in un volume tale da sommergere l&#39;intera membrana, per esempio, con rapporto di diluizione 1: 1.000, aggiungere 1 ml di anticorpo (concentrazione magazzino: 1 mg / ml) a 999 ml di PBS per una miscela totale 1 ml. </li><li> Lavare la membrana con 5 ml di PBST (PBS con 0,1% Tween20). </li><li> Incubare con anticorpo secondario coniugato con rafano perossidasi (HRP) con diluizioni in PBS a 1: 5.000 a temperatura ambiente per 1 ora con dondolo. Preparare una miscela in un volume tale da sommergere l&#39;intera membrana, per esempio, con rapporto di diluizione 1: 5.000, aggiungere 1 ml di anticorpo (concentrazione magazzino: 1 mg / ml) a 4,999 microlitri PBS per una miscela totale 5 ml. </li><li> Lavare la membrana con 5 ml di PBST e ripetere due volte. </li><li> Applicare il substrato chemiluminescente per la membrana con un volume per coprire la membrana e incubare seguendo le istruzioni del substrato utilizzato. NOTA: La sensibilità del substrato di rilevamento proteina può variare, si consiglia un substrato con la massima sensibilità per i segnali molto bassi in caso crescente concentrazione di anticorpi non aumenta i segnali. </li><li> Cattura i segnali chemiluminescenti utilizzando un sensore basato su telecamera CCD. Regolare il rapporto di diluizione anticorpo primario e secondario se i segnali sono deboli e non specifici. Incubare più nel bloccare soluzione se il segnale di fondo è alto. </li></ol>

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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Designing+ECM-mimetic+materials+using+protein+engineering.">Designing ECM-mimetic materials using protein engineering.</a> Acta Biomater. 10 (4), 1751-1760 (2014).</li><li>Annabi, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elastomeric+recombinant+protein-based+biomaterials.">Elastomeric recombinant protein-based biomaterials.</a> Biochemical Engineering Journal. 77, 110-118 (2013).</li><li>Heilshorn, S. C., DiZio, K. A., Welsh, E. R., Tirrell, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cell+adhesion+to+the+fibronectin+CS5+domain+in+artificial+extracellular+matrix+proteins.">Endothelial cell adhesion to the fibronectin CS5 domain in artificial extracellular matrix proteins.</a> Biomaterials. 24 (23), 4245-4252 (2003).</li><li>Simnick, A. J., Lim, D. W., Chow, D., Chilkoti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomedical+and+Biotechnological+Applications+of+Elastin-Like+Polypeptides.">Biomedical and Biotechnological Applications of Elastin-Like Polypeptides.</a> Polymer Reviews. 47 (1), 121-154 (2007).</li><li>Tjin, M. S., Chua, A. W. C., Ma, D. R., Lee, S. T., Fong, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Epidermal+Keratinocyte+Cell+Response+on+Integrin-Specific+Artificial+Extracellular+Matrix+Proteins.">Human Epidermal Keratinocyte Cell Response on Integrin-Specific Artificial Extracellular Matrix Proteins.</a> Macromolecular Bioscience. 14 (8), 1125-1134 (2014).</li><li>MacNeil, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+and+opportunities+for+tissue-engineered+skin.">Progress and opportunities for tissue-engineered skin.</a> Nature. 445 (7130), 874-880 (2007).</li><li>Kariya, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+regulation+of+cellular+adhesion+and+migration+by+recombinant+laminin-5+forms+with+partial+deletion+or+mutation+within+the+G3+domain+of+&#945;3+chain.">Differential regulation of cellular adhesion and migration by recombinant laminin-5 forms with partial deletion or mutation within the G3 domain of &#945;3 chain.</a> J Cell Biochem. 88 (3), 506-520 (2003).</li><li>Shang, M., Koshikawa, N., Schenk, S., Quaranta, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+LG3+module+of+laminin-5+harbors+a+binding+site+for+integrin+&#945;3&#914;1+that+promotes+cell+adhesion,+spreading,+and+migration.">The LG3 module of laminin-5 harbors a binding site for integrin &#945;3&#914;1 that promotes cell adhesion, spreading, and migration.</a> J Biol Chem. 276 (35), 33045-33053 (2001).</li><li>Hassouneh, W., Christensen, T., Chilkoti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elastin-like+polypeptides+as+a+purification+tag+for+recombinant+proteins.">Elastin-like polypeptides as a purification tag for recombinant proteins.</a> Current Protocols in Protein Science. , 6.11.1-6.11.16 (2010).</li><li>Keeley, F., Mecham, R., Keeley, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch.+4.">Ch. 4.</a> Evolution of Extracellular Matrix.Biology of Extracellular Matrix. , 73-119 (2013).</li><li>Le, D. H. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-Assembly+of+Elastin-Mimetic+Double+Hydrophobic+Polypeptides.">Self-Assembly of Elastin-Mimetic Double Hydrophobic Polypeptides.</a> Biomacromolecules. 14 (4), 1028-1034 (2013).</li><li>MacEwan, S. R., Hassouneh, W., Chilkoti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-chromatographic+Purification+of+Recombinant+Elastin-like+Polypeptides+and+their+Fusions+with+Peptides+and+Proteins+from+Escherichia+coli.">Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli.</a> Journal of visualized experiments: JoVE. (88), e51583 (2014).</li><li>Meyer, D. E., Chilkoti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+encoded+synthesis+of+protein-based+polymers+with+precisely+specified+molecular+weight+and+sequence+by+recursive+directional+ligation:+examples+from+the+elastin-like+polypeptide+system.">Genetically encoded synthesis of protein-based polymers with precisely specified molecular weight and sequence by recursive directional ligation: examples from the elastin-like polypeptide system.</a> Biomacromolecules. 3 (2), 357-367 (2002).</li></ol>

Gli autori dichiarano di non avere interessi finanziari in competizione.

Ingegneria proteica ricombinante è una tecnica versatile per creare sostanze proteiche nuovi con un approccio bottom-up. I materiali a base di proteine ​​può essere progettato per avere funzionalità multiple, su misura secondo l&#39;applicazione di interesse. A causa della crescente avanzamento nelle tecnologie di clonaggio e di espressione proteica, è diventato relativamente semplice (ed economico) per creare una varietà di proteine ​​artificiali in modo riproducibile e scalabile. Il dominio elastina-like ...

For several decades, both synthetic and natural materials have been explored for use as scaffolds in tissue engineering1,2. While synthetic materials such as polymers offer excellent structural integrity and tunable mechanical properties, they often have insufficient bioactivity to promote growth and infiltration of tissues. On the other hand, natural materials such as extracellular matrix (ECM) proteins have excellent biological activity, but have limitations such as batch-to-batch variability, rapid degradation and immunogenicity issues. As such, recombinant proteins are desired, since they can be designed to mimic only the desirable properties of native proteins3,4.
Recombinant protein engineering has garnered widespread interests as a versatile platform for the design and production of novel artificial protein biopolymers. By controlling the genetic sequence, the functionalities of the artificial proteins can be tailored for a wide variety of applications5,6. In particular, artificial extracellular matrix (aECM) proteins can be tailored to have multiple functionalities for applications in tissue engineering, regeneration and wound repair2,7. More importantly, advances in cloning and purification technologies have increased scalability and reduced the cost of manufacturing recombinant proteins tremendously. It is possible to produce large quantities of recombinant proteins at low production costs which are economic for use in the clinic5. 
Artificial extracellular matrix proteins have been developed for tissue engineering applications8-11. For instance, Tirrell et al. designed a small diameter vascular graft using artificial proteins containing fibronectin CS5 sequence and elastin-like repeats (ELP-CS5). They showed that human umbilical vein endothelial cells (HUVECs) were able to adhere and grow on these materials12. Others have also incorporated short bioactive sequences taken from fibronectin, collagen, laminin, fibrinogen and vitronectin as well as structural domains that mimic elastin, spider silk and collagens to create a variety of fusion proteins10. Bulk cross-linked films made out of elastin-based aECM proteins also exhibited mechanical properties similar to that of native elastin (elastic moduli ranges between 0.3-0.6 MPa)13. Subsequently, aECM proteins containing longer fibronectin fragments were also reported to accelerate wound healing in vitro due to increased integrin binding affinities8. 
Recently, integrin-specific artificial ECM proteins have been developed by Tjin and coworkers14. Each aECM protein contains a bioactive cell-binding domain taken from ECM components of native human skin2,7,15, such as laminin-5, collagen-IV and fibronectin. For example, the integrin &#945;3&#914;1 has been shown to bind the PPFLMLLKGSTR sequence found in the laminin-5 alpha-3 chain globular domain 3 (LG3)16,17. In their report, they showed that primary human skin epidermal keratinocytes preferentially engage different integrins for binding to each of the aECM proteins, depending on the type of cell binding domain present.
The aECM proteins discussed in the work by Tjin et al. contain flanking elastin-like domains {(VPGIG)2VPGKG(VPGIG)2}8 that confer elasticity which mimics the mechanical properties of human skin. In addition, the incorporation of lysine residues within the elastin-like repeats also increases the overall protein solubility in aqueous solvents. In addition, the lysine residues also serve as crosslinking sites to facilitate the formation of crosslinked aECM films12. Inclusion of elastin-like repeats within the aECM protein sequence allow the proteins to be readily purified via Inverse Transition Cycling (ITC)14. Elastins undergo a sharp and reversible phase transition at a specific temperature known as the lower critical solution temperature (LCST) or the inverse transition temperature (Tt)18-20. Elastins and elastin-like repeats adopt hydrophilic random coil conformations below their LCST and become soluble in water, whereas above their LCST, elastins aggregate rapidly into micron-size particles. Such phase transitions are reversible and hence, can be exploited to allow elastin-based aECM proteins to be readily purified via the ITC technique21.
In this work, we report a generalized procedure to design, construct and purify artificial ECM proteins containing bioactive cell-binding domains, fused to elastin-like repeats. The process to design and clone the plasmids that encode for the amino acid sequences for the aECM proteins is described. The steps involved to purify the aECM proteins using ITC are outlined. Finally, the methods to determine the purity of the aECM proteins obtained using SDS-PAGE electrophoresis and Western Blotting are discussed.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>pET22b (+)</td>
			<td>Novagen</td>
			<td>69744</td>
			<td>T7 expression vectors with resistance to ampicillin&#160;</td>
		</tr>
		<tr>
			<td>BL21(DE3)pLysS&#160;</td>
			<td>Invitrogen</td>
			<td>C6060-03</td>
			<td>additional antibiotics - chloramphenicol</td>
		</tr>
		<tr>
			<td>Isopropyl-beta-D-thiogalactoside (IPTG)</td>
			<td>Gold Biotechnology</td>
			<td>I2481C</td>
			<td>1M stock solution with autoclaved water, make fresh prior to induction.</td>
		</tr>
		<tr>
			<td>QIAprep Spin Miniprep Kit</td>
			<td>Qiagen</td>
			<td>27106</td>
			<td>plasmid isolation kit</td>
		</tr>
		<tr>
			<td>T4 ligase</td>
			<td>New England Biolabs</td>
			<td>M0202S</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Affymetrix</td>
			<td>11259</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloramphenicol</td>
			<td>Affymetrix</td>
			<td>23660</td>
			<td></td>
		</tr>
		<tr>
			<td>Zymoclean&#8482; gel DNA recovery kit</td>
			<td>Zymo Research</td>
			<td>D4001</td>
			<td></td>
		</tr>
		<tr>
			<td>XL10-gold strain</td>
			<td>Agilent Technologies</td>
			<td>200315</td>
			<td></td>
		</tr>
	</tbody>
</table>

design and construction of artificial extracellular matrix (aecm) proteins from escherichia coli for skin tissue engineering

Recombinant technology is a versatile platform to create novel artificial proteins with tunable properties. For the last decade, many artificial proteins that have incorporated functional domains derived from nature (or created de novo) have been reported. In particular, artificial extracellular matrix (aECM) proteins have been developed; these aECM proteins consist of biological domains taken from fibronectin, laminins and collagens and are combined with structural domains including elastin-like repeats, silk and collagen repeats. To date, aECM proteins have been widely investigated for applications in tissue engineering and wound repair. Recently, Tjin and coworkers developed integrin-specific aECM proteins designed for promoting human skin keratinocyte attachment and propagation. In their work, the aECM proteins incorporate cell binding domains taken from fibronectin, laminin-5 and collagen IV, as well as flanking elastin-like repeats. They demonstrated that the aECM proteins developed in their work were promising candidates for use as substrates in artificial skin. Here, we outline the design and construction of such aECM proteins as well as their purification process using the thermo-responsive characteristics of elastin.

Nella progettazione di proteine ​​di fusione contenenti ripetizioni elastina simile, è importante mantenere un contenuto di elastina generale, abbastanza grande frazione della proteina di fusione 18. Questo per garantire che il costrutto proteina di fusione mantiene le sue caratteristiche elastina simili, al fine di utilizzare ITC per la purificazione. Il design e le proteine ​​sequenze descritte in questa sezione AECM particolare sono stati presi dal lavoro di Tjin et al. 14. In ...

Watch this Scientific Journal Video about Design and Construction of Artificial Extracellular Matrix aECM Proteins from Escherichia coli for Skin Tissue Engineering at JoVE.com

Design and Construction of Artificial Extracellular Matrix aECM Proteins from Escherichia coli for Skin Tissue Engineering

Recombinant technologies have enabled material designers to create novel artificial proteins with customized functionalities for tissue engineering applications. For example, artificial extracellular matrix proteins can be designed to incorporate structural and biological domains derived from native ECMs. Here, we describe the construction and purification of aECM proteins containing elastin-like repeats.

Progettazione e Costruzione di Artificiale matrice extracellulare (AECM) Proteine ​​da<em&gt; Escherichia coli</em&gt; Per la pelle Ingegneria dei Tessuti

Recombinant technology is a versatile platform to create novel artificial proteins with tunable properties. For the last decade, many artificial proteins ...

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Research

JoVE Journal

Bioengineering

Design and Construction of Artificial Extracellular Matrix (aECM) Proteins from Escherichia coli for Skin Tissue Engineering

8.8K Views.  Nanyang Technological University. Recombinant technologies have enabled material designers to create novel artificial proteins with customized functionalities for tissue engineering applications. For example, artificial extracellular matrix proteins can be designed to incorporate structural and biological domains derived from native ECMs. Here, we describe the construction and purification of aECM proteins containing elastin-like repeats. 

Video: Design and Construction of Artificial Extracellular Matrix aECM Proteins from Escherichia coli for Skin Tissue Engineering

Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, currently available routine methods require processing steps and alter sample integrity. Raman spectroscopy is a fast method that enables the measurement of biological samples without the need for further processing steps. This laser-based technology detects the inelastic scattering of monochromatic light.6 As every chemical vibration is assigned to a specific Raman band (wavenumber in cm-1), each biological sample features a typical spectral pattern due to their inherent biochemical composition.7-9 Within Raman spectra, the peak intensities correlate with the amount of the present molecular bonds.1 Similarities and differences of the spectral data sets can be detected by employing a multivariate analysis (e.g. principal component analysis (PCA)).10
Here, we perform Raman spectroscopy of living cells and native tissues. Cells are either seeded on glass bottom dishes or kept in suspension under normal cell culture conditions (37 &deg;C, 5% CO2) before measurement. Native tissues are dissected and stored in phosphate buffered saline (PBS) at 4 &deg;C prior measurements. Depending on our experimental set up, we then either focused on the cell nucleus or extracellular matrix (ECM) proteins such as elastin and collagen. For all studies, a minimum of 30 cells or 30 random points of interest within the ECM are measured. Data processing steps included background subtraction and normalization.

Raman spectroscopy is a suitable technique for the non-contact, label-free analysis of living cells, tissue-engineered constructs and native tissues. Source-specific spectral fingerprints can be generated and analyzed using multivariate analysis.

Senza contatto, Label-free monitoraggio delle cellule e matrice extracellulare mediante Spettroscopia Raman

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, ...

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Bioingegneria

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

We designed a loading device that is capable of applying uniaxial or biaxial mechanical strain to a tissue engineered biocomposites fabricated for transplantation. While the device primarily functions as a bioreactor that mimics the native mechanical strains, it is also outfitted with a load cell for providing force feedback or mechanical testing of the constructs. The device subjects engineered cartilage constructs to biaxial mechanical loading with great precision of loading dose (amplitude and frequency) and is compact enough to fit inside a standard tissue culture incubator. It loads samples directly in a tissue culture plate, and multiple plate sizes are compatible with the system. The device has been designed using components manufactured for precision-guided laser applications. Bi-axial loading is accomplished by two orthogonal stages. The stages have a 50 mm travel range and are driven independently by stepper motor actuators, controlled by a closed-loop stepper motor driver that features micro-stepping capabilities, enabling step sizes of less than 50 nm. A polysulfone loading platen is coupled to the bi-axial moving platform. Movements of the stages are controlled by Thor-labs Advanced Positioning Technology (APT) software. The stepper motor driver is used with the software to adjust load parameters of frequency and amplitude of both shear and compression independently and simultaneously. Positional feedback is provided by linear optical encoders that have a bidirectional repeatability of 0.1 &mu;m and a resolution of 20 nm, translating to a positional accuracy of less than 3 &mu;m over the full 50 mm of travel. These encoders provide the necessary position feedback to the drive electronics to ensure true nanopositioning capabilities. In order to provide the force feedback to detect contact and evaluate loading responses, a precision miniature load cell is positioned between the loading platen and the moving platform. The load cell has high accuracies of 0.15% to 0.25% full scale.

We designed a novel mechanical loading bioreactor that can apply uniaxial or biaxial mechanical strain to a cartilage biocomposite prior to transplantation into an articular cartilage defect.

Progettazione di un biassiale Carico meccanico bioreattore per l&#39;ingegneria tissutale

We  designed a loading device that is capable of applying uniaxial or biaxial  mechanical strain to a tissue engineered biocomposites fabricated for  ...

Tissue Engineering: Construction of a Multicellular 3D Scaffold for the Delivery of Layered Cell Sheets

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to assist the body in recovery and repair. For example, TE approaches may be able to attenuate heart remodeling after myocardial infarction (MI) and possibly increase total heart function to a near normal pre-MI level.4 As with any functional tissue, successful regeneration of cardiac tissue involves the proper delivery of multiple cell types with environmental cues favoring integration and survival of the implanted cell/tissue graft. Engineered tissues should address multiple parameters including: soluble signals, cell-to-cell interactions, and matrix materials evaluated as delivery vehicles, their effects on cell survival, material strength, and facilitation of cell-to-tissue organization. Studies employing the direct injection of graft cells only ignore these essential elements.2,5,6 A tissue design combining these ingredients has yet to be developed. Here, we present an example of integrated designs using layering of patterned cell sheets with two distinct types of biological-derived materials containing the target organ cell type and endothelial cells for enhancing new vessels formation in the &#8220;tissue&#8221;. Although these studies focus on the generation of heart-like tissue, this tissue design can be applied to many organs other than heart with minimal design and material changes, and is meant to be an off-the-shelf product for regenerative therapies. The protocol contains five detailed steps. A temperature sensitive Poly(N-isopropylacrylamide) (pNIPAAM) is used to coat tissue culture dishes. Then, tissue specific cells are cultured on the surface of the coated plates/micropattern surfaces to form cell sheets with strong lateral adhesions. Thirdly, a base matrix is created for the tissue by combining porous matrix with neovascular permissive hydrogels and endothelial cells. Finally, the cell sheets are lifted from the pNIPAAM coated dishes and transferred to the base element, making the complete construct.

For creation of highly organized structures of complex tissue, one must assemble multiple material and cell types into an integrated composite. This combinatorial design incorporates organ-specific layered cell sheets with two distinct biologically-derived materials containing a strong fibrous matrix base, and endothelial cells for enhancing new vessels formation.

Ingegneria dei tessuti: Costruzione di un multicellulare 3D Ponteggio per la consegna di Layered fogli Cellulari

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

Generazione di una tridimensionale a tutto spessore della pelle Equivalent e Automated Ferimento

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Epithelial Cell Repopulation and Preparation of Rodent Extracellular Matrix Scaffolds for Renal Tissue Development

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model substrates for organ-scale tissue development. Sprague Dawley rat kidneys are cannulated by inserting a catheter into the renal artery and perfused with a series of low-concentration detergents (Triton X-100 and sodium dodecyl sulfate (SDS)) over 26 hr to derive intact, whole-kidney scaffolds with intact perfusable vasculature, glomeruli, and renal tubules. Following decellularization, the renal scaffold is placed inside a custom-designed perfusion bioreactor vessel, and the catheterized renal artery is connected to a perfusion circuit consisting of: a peristaltic pump; tubing; and optional probes for pH, dissolved oxygen, and pressure. After sterilizing the scaffold with peracetic acid and ethanol, and balancing the pH (7.4), the kidney scaffold is prepared for seeding via perfusion of culture medium within a large-capacity incubator maintained at 37 &#176;C and 5% CO2. Forty million renal cortical tubular epithelial (RCTE) cells are injected through the renal artery, and rapidly perfused through the scaffold under high flow (25 ml/min) and pressure (~230 mmHg) for 15 min before reducing the flow to a physiological rate (4 ml/min). RCTE cells primarily populate the tubular ECM niche within the renal cortex, proliferate, and form tubular epithelial structures over seven days of perfusion culture. A 44 &#181;M resazurin solution in culture medium is perfused through the kidney for 1 hr during medium exchanges to provide a fluorometric, redox-based metabolic assessment of cell viability and proliferation during tubulogenesis. The kidney perfusion bioreactor permits non-invasive sampling of medium for biochemical assessment, and multiple inlet ports allow alternative retrograde seeding through the renal vein or ureter. These protocols can be used to recellularize kidney scaffolds with a variety of cell types, including vascular endothelial, tubular epithelial, and stromal fibroblasts, for rapid evaluation within this system.

This protocol describes decellularization of Sprague Dawley rat kidneys by antegrade perfusion of detergents through the vasculature, producing acellular renal extracellular matrices that serve as templates for repopulation with human renal epithelial cells. Recellularization and use of the resazurin perfusion assay to monitor growth is performed within specially-designed perfusion bioreactors.

Ripopolamento cellule epiteliali e preparazione di roditori matrice extracellulare Ponteggi per lo sviluppo del tessuto renale

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

Tessuti epiteliali Ingegneria tridimensionali integrati all&#39;interno di matrice extracellulare

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

Preparation of Extracellular Matrix Protein Fibers for Brillouin Spectroscopy

Brillouin spectroscopy is an emerging technique in the biomedical field. It probes the mechanical properties of a sample through the interaction of visible light with thermally induced acoustic waves or phonons propagating at a speed of a few km/sec. Information on the elasticity and structure of the material is obtained in a nondestructive contactless manner, hence opening the way to in vivo applications and potential diagnosis of pathology. This work describes the application of Brillouin spectroscopy to the study of biomechanics in elastin and trypsin-digested type I collagen fibers of the extracellular matrix. Fibrous proteins of the extracellular matrix are the building blocks of biological tissues and investigating their mechanical and physical behavior is key to establishing structure-function relationships in normal tissues and the changes which occur in disease. The procedures of sample preparation followed by measurement of Brillouin spectra using a reflective substrate are presented together with details of the optical system and methods of spectral data analysis.

We present a protocol for the application of Brillouin light scattering spectroscopy to elastin and trypsin-purified type I collagen fibers of the extracellular matrix to extract their full elastic properties.

Preparazione della matrice extracellulare fibre proteiche per Brillouin Spettroscopia

Brillouin spectroscopy is an emerging technique in the biomedical field. It probes the mechanical properties of a sample through the interaction of ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

Sintonizzabile idrogel da polmonare matrice extracellulare per colture cellulari 3D

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus of this article is on the methods for fabricating ECM-derived porous foams and microcarriers for use as biologically-relevant substrates in advanced 3D in vitro cell culture models or as pro-regenerative scaffolds and cell delivery systems for tissue engineering and regenerative medicine. Using decellularized tissues or purified insoluble collagen as a starting material, the techniques can be applied to synthesize a broad array of tissue-specific bioscaffolds with customizable geometries. The approach involves mechanical processing and mild enzymatic digestion to yield an ECM suspension that is used to fabricate the three-dimensional foams or microcarriers through controlled freezing and lyophilization procedures. These pure ECM-derived scaffolds are highly porous, yet stable without the need for chemical crosslinking agents or other additives that may negatively impact cell function. The scaffold properties can be tuned to some extent by varying factors such as the ECM suspension concentration, mechanical processing methods, or synthesis conditions. In general, the scaffolds are robust and easy to handle, and can be processed as tissues for most standard biological assays, providing a versatile and user-friendly 3D cell culture platform that mimics the native ECM composition. Overall, these straightforward methods for fabricating customized ECM-derived foams and microcarriers may be of interest to both biologists and biomedical engineers as tissue-specific cell-instructive platforms for in vitro and in vivo applications.

The tissue-specific extracellular matrix (ECM) is a key mediator of cell function. This article describes methods for synthesizing pure ECM-derived foams and microcarriers that are stable in culture without the need for chemical crosslinking for applications in advanced 3D in vitro cell culture models or as pro-regenerative bioscaffolds.

Fabbricazione di Schiume matrice extracellulare-derivati ​​e microcarriers come tessuto-specifica delle cellule, Cultura e piattaforme di distribuzione

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus ...

Fabricating a Kidney Cortex Extracellular Matrix-Derived Hydrogel

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust mechanical support for in vitro cell culture but lack the necessary protein and ligand composition to elicit physiological behavior from cells. This manuscript describes a fabrication method for a kidney cortex ECM-derived hydrogel with proper mechanical robustness and supportive biochemical composition. The hydrogel is fabricated by mechanically homogenizing and solubilizing decellularized human kidney cortex ECM. The matrix preserves native kidney cortex ECM protein ratios while also enabling gelation to physiological mechanical stiffnesses. The hydrogel serves as a substrate upon which kidney cortex-derived cells can be maintained under physiological conditions. Furthermore, the hydrogel composition can be manipulated to model a diseased environment which enables the future study of kidney diseases.

Here we present a protocol to fabricate a kidney cortex extracellular matrix-derived hydrogel to retain the native kidney extracellular matrix (ECM) structural and biochemical composition. The fabrication process and its applications are described. Finally, a perspective on using this hydrogel to support kidney-specific cellular and tissue regeneration and bioengineering is discussed.

Fabbricando un rene corteccia extracellulare Matrix-derivato idrogel

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust ...

Progettazione e Costruzione di Artificiale matrice extracellulare (AECM) Proteine ​​da

Progettazione e Costruzione di Artificiale matrice extracellulare (AECM) Proteine da